Project Summary During early embryogenesis, pluripotent cells specify their fates by integrating multiple signals delivered at different times, places, and with different dynamics. In the mouse embryo, interplay between 3 signaling pathways - BMP4, Wnt, and Nodal - initiate the induction and patterning of embryonic germ layers. However, the relevance of these pathways to human development is not understood. Furthermore, how multiple dynamic signaling pathways are integrated to define cellular fate is unclear. Here we propose to use human embryonic stem cells (hESCs) to understand how individual cells process these dynamic signals to generate discrete fates. To address this, we developed 2 innovative technologies. First, microfluidics to precisely control the timing of ligand application and follow the behavior of the signal transducer SMAD4, with which we demonstrated that TGF? signaling was adaptive. Second, micropatterns to control hESC colony size and geometry, to show that in response to BMP4, hESCs cultured in circular colonies self-organize into radially symmetric patterns of discrete embryonic germ layers. This remarkably recapitulates the proximal-distal axis of the gastrulating mouse embryo. In this competitive renewal, we combine the strengths of both technologies to deliver distinct dynamics of ligand presentation by microfluidics to CRISPR-edited hESC lines cultured in micropatterned colonies. Four independent signaling-reporter hESC lines that fluorescently tag SMAD1, SMAD2, SMAD4, and ?-CATENIN will be used to visualize signaling, and one triple-tagged, fate-reporter CRISPR-edited line that fluorescently tags SOX2, BRACHYURY, and SOX17, will be used to monitor fate acquisition. Our CRISPR-reporter lines will be used to measure signaling dynamics and fate acquisition, with single cell resolution and in real-time by video-microscopy, when cells are presented with BMP4, Wnt3A, and Activin/Nodal either as a persistent step of defined concentration, or as one of defined duration. We propose three specific aims. In aim1, the three ligands will be presented to our signaling-reporter CRISPR lines, to follow the behavior of the four tagged signal transducers and to determine the dynamic behavior of each pathway. In aim2, using the same approach, we will evaluate pathway output by measuring the activity of transcriptional reporters for the three ligands, and use our triple fate-reporter CRISPR line to follow fate acquisition. These two approaches will establish a quantitative link between signaling dynamics, transcriptional output, and fate determination. In aim3, large datasets obtained from aims1 and 2, will be used to model the kinetics of signal transduction, and provide a mathematical paradigm to explain hESC self-organization. The resolution of our three aims will have a strong impact on our understanding of the dynamic integration of signaling pathways underlying human cell fate specification with direct relevance to both basic understanding, and clinical applications of hESCs.